Compositions comprising and methods of using norfluoxetine

ABSTRACT

The invention encompasses methods of using racemic and enantiomerically pure norfluoxetine, as well as pharmaceutical compositions, pharmaceutical unit dosage forms, and kits comprising racemic and enantiomerically pure norfluoxetine. In particular, the invention encompasses a method of inhibiting or reducing the production of PGE 2 . The invention further encompasses a method of treating or preventing inflammation in a patient, as well as a method of treating or preventing a disease or disorder such as, but not limited to, autoimmune diseases, arthritis, neurologic diseases, inflammatory diseases, fibromyalgia, pain resulting from inflammation, neuropathic pain, and cancer.

1. FIELD OF THE INVENTION

This invention relates to pharmaceutical compositions comprisingenantiomerically pure R— or S-norfluoxetine, and methods of usingenantiomerically pure R— or S-norfluoxetine for the treatment orprevention of inflammation and related disorders.

2. BACKGROUND OF THE INVENTION 2.1. Inflammation

Inflammation can be elicited by a variety of stimuli. Examples includeinfectious agents, ischemia, antigen-antibody interactions, and thermalor other physical injury. Goodman and Gilman's The Pharmcological Basisof Therapeutics 617-618 (9^(th) ed.; 1996). Consequently, inflammationis associated with a large number of diseases and conditions.

For example, the inflammation of connective tissue is characteristic ofdiseases and disorders such as rheumatoid arthritis, tendinitis, andtenosynovitis, which affect tens of thousands of people each year. MerckManual 416-422, 455, 479-481 (17^(th) ed.; 1999). Other examples ofinflammation include chronic inflammation of the gastrointestinal tract,which is characteristic of diseases such as Crohn's disease andulcerative colitis (id. at 302); inflammation of nerve tissue, which ischaracteristic of neuropathies such as Guillain-Barré syndrome, (id. at1494-1495); and inflammation of blood vessels, which is characteristicof vasculitis (e.g., Henoch-Schönlein syndrome, or Pseudomonassepticemia or drug-induced vasculitis, erythema nodosum, polyarteritisnodosa, temporal arteritis, and Takayasu's arteritis). Id. at 437-442. Asignificant number of people also suffer from systemic inflammation,which is typical of diseases such as Sjögren's Disorder, Behcet'sSyndrom, relapsing polychondritis, systemic lupus erythematosus,eosinophilic fasciitis, polymyositis, and dermatomyositis. Id. at423-436. Other examples and types of inflammation are well known to themedical community.

A variety of drugs can be used to treat inflammation, some of which workbetter than others, and some of which exhibit fewer or less severeadverse effects than others. But the safety and effectiveness of aparticular anti-inflammatory drug can depend on a variety of factors,including its mechanism of action.

The inflammatory process is a complex and varied one. For example,several classes of leukocytes reportedly play essential roles ininflammation, as do various molecules that adhere leukocytes to sites ofinflammation. It has been suggested that non-steroidal anti-inflammatorydrugs (NSAIDs) act by inhibiting the expression or activity of certainadhesion molecules, although the principle effects of NSAIDs arereportedly due to their inhibition of prostaglandin biosynthesis.Goodman and Gilman's The Pharmcological Basis of Therapeutics 618,620-622 (9^(th) ed.; 1996). A large number of soluble mediators (e.g.,histamine) also appear to be involved in the inflammation process, as doseveral different cytokines such as Interleukin 1 (IL-1) and TumorNecrosis Factor (TNF). Id. Other cytokines and growth factors that maybe involved in the process include IL-2, IL-6, IL-8, and GM-CSF.Peptides that promote firing of pain fibers, such as Substance P, havealso been implicated in the process. Id. at 619. In addition to theseand the myriad of other compounds, processes, and interactionsassociated with inflammation, is has been suggested that bradykinin and5-hydroxytryptamine (serotonin, 5-HT) may play a role in mediatingcertain types of inflammation. Id.

Because inflammation is a symptom common to so many diseases anddisorders, there remains a need for new anti-inflammatory and analgesicdrugs. A particular need exists for anti-inflammatory drugs that operatemore effectively or incur fewer adverse effects than those currentlyavailable. For example, NSAIDs can cause the formation of gastric orintestinal ulcerations, can inhibit platelet aggregation, and canpromote the retention of salt and water, thereby reducing theeffectiveness of antihypertensive regimes. Id. at 622-623.

It is also desirable to provide new anti-inflammatory drugs that incurfewer or different adverse drug-drug interactions than those which arecurrently available. For example, when NSAIDs are administered withdiuretics such as hydrochlorothiazide, they can increase the risk ofrenal failure. See, e.g., Physicians' Desk Reference 3287 (54^(th) ed.;2000). NSAIDs such as ketoprofen can also affect the elimination ofcertain drugs (e.g., methotrexate), leading to elevated serum levels ofthose drugs and increasing their toxicity. Id. See also, Goodman andGilman's The Pharmcological Basis of Therapeutics 624-625 (9^(th) ed.;1996).

Perhaps for these reasons; researchers have investigated whether drugstypically not associated with the treatment of inflammation can affectit. For example, it has been reported that certain psychotropic agentsinhibit the conversion of arachidonic acid to Prostaglandin E₂ (PGE₂) invitro. Krupp, P., and West, M. Experientia 31:330-331 (1975). It hasalso been reported that certain tricyclic antidepressants and methylxanthine compounds behave as prostaglandin antagonists in vitro.Horrobin, D. F., and Manku, M. S., Med. Hypothesis 3:71-86 (1977).Studies done with rats, however, have suggested alternative mechanismsby which specific anti-depressants may affect pain and inflammation.See, e.g., El-Mahdy, S. A. M., et al., J. Pharm. Pharmacol. 42:522-524(1990). See also, Michelson, D., et al., Agents Actions 42:25-28 (1994);Butler, S. H., et al., Pain 23:159-175 (1985); Bianci, M., et al., Eur.J. Pharmacol. 219:113-116 (1992); Martelli, E. A., et al., Eur. J.Pharmacol. 2:229-233 (1967);

Research has shown quite clearly that different drugs act in differentways. For example, tricyclic antidepressants reportedly affectpolymorphonuclear cell (PMN) migration, an activity associated with therecruitment of leukocytes to loci of inflammation or infection.Sacerdote, P., et al., Gen. Pharmac. 25(3):409-412 (1994). However, theanti-depressant fluoxetine reportedly does not affect PMN migration. Id.at 411. The different activities of fluoxetine and tricyclicantidepressants has also been reported by Bianchi and coworkers. See,e.g., Bianchi, M., and Panerai, A. E., Pharmacol. Res. 33(4/5):235-238(1996). Further research has shown that the antinociceptive action oftricyclic antidepressants is independent of the anti-depressant effect.See, e.g., Bianchi, M., et al., Pharm. Biochem. Behav. 48(4):1037-1040(1994). It has also been reported that the effects that anti-depressantshave on mechanisms associated with pain and inflammation vary betweenspecies. See, e.g., Ventafridda, V., et al., Pain 43:155-162 (1990). Forexample, drugs can affect rats and humans differently. Id.

2.2. Fluoxetine and Norfluoxetine

Fluoxetine, which is chemically named(±)-N-methyl-γ-[4-(trifluoromethyl)-phenoxy]benzenepropanamine, is ananti-depressant that is sold under the trade name Prozac®. Physicians'Desk Reference 962-966 (54^(th) ed.; 2000). See also U.S. Pat. Nos.4,018,895 and 4,194,009, each of which is incorporated herein byreference.

Although racemic fluoxetine is approved as being safe and effective onlyfor the treatment of depression in the United States, its effect onother diseases and conditions has been investigated. One example isfibromyalgia. Goldenberg, D., et al., Arthritis & Rheumatism39(11):1852-1859 (1996). Reports also suggest that racemic fluoxetinecan affect inflammatory edema in the paws of rats, as well assubcutaneous carrageenin-induced inflammation in rats. Bianchi, M., etal., Eur. J. Pharmacol. 263:81-84 (1994); Bianchi, M., et al., Inflamm.Res. 44:466-469 (1995). However, the mechanism by which racemicfluoxetine acts to reduce inflammation in rats remains unclear. See,e.g., Yaron, I., et al., Arthritis & Rheumatism 42(12):2561-2568 (1999).It has also been reported that the combined administration of racemicfluoxetine and codeine or dextropropoxyphene can be used to treat pain.See U.S. Pat. Nos. 4,683,235 and 4,594,358 and European PatentApplication 0193355.

Fluoxetine is commercially available as a racemic compound. It has beenreported, however, that enantiomerically pure enantiomers of fluoxetine(i.e., R(−)-fluoxetine and S(+)-fluoxetine) are reportedly useful in thetreatment of depression and other conditions, such as pain, in humans.See, e.g., U.S. Pat. Nos. 5,708,035, 5,648,396, and 5,589,511.

The primary metabolite of fluoxetine in humans in norfluoxetine, whichis described in U.S. Pat. No. 4,313,896 and European Patent Application0 369 685. Norfluoxetine, which is chemically named(±)-γ-[4-(trifluoromethyl)-phenoxy]benzenepropanamine, is a racemicmixture of two enantiomers, R-norfluoxetine and S-norfluoxetine, thestructures of which are shown below:

It has been reported that racemic norfluoxetine can be used in thetreatment of disorders such as depression, abnormal muscular function,abnormal pituitary function, inability to sleep, abnormal sexualperformance, and abnormal appetite (U.S. Pat. No. 4,313,896),hypertension (U.S. Pat. No. 4,329,356), atherosclerosis (U.S. Pat. No.4,444,178), anxiety (U.S. Pat. No. 4,590,213), weight gain (U.S. Pat.No. 4,895,845), and diabetes (EPA 0 294 028). Some animal studies havealso suggested that racemic fluoxetine has analgesic activity. Benfield,P., et al., Drugs 32:481-508, 489 (1986); Robertson, D. W., et al., J.Med. Chem. 31:1412-1417 (1988). It has also been reported that thecombined administration of racemic norfluoxetine and codeine ordextropropoxyphene can be used to treat pain. See U.S. Pat. Nos.4,683,235 and 4,594,358 and European Patent Application 0193355.

Although uses have been suggested for racemic norfluoxetine, only theS-enantiomer exhibits the potent and selective serotonin uptakeinhibition of its racemic parent. R-norfluoxetine is significantly lesspotent than racemic fluoxetine in inhibiting serotonin uptake.Physicians' Desk Reference 962 (54^(th) ed.; 2000).

3. SUMMARY OF THE INVENTION

This invention is based, in part, on the unexpected discovery thatmetabolites of serotonin reuptake inhibitors can exhibitanti-inflammatory activity, and can be used in the treatment orprevention of a large number of diseases and disorders. The inventionencompasses methods of treating or preventing disease and disorderswhich include, but are not limited to, inflammation, autoimmune diseases(e.g. lupus erythematosus and multiple sclerosis), arthritis (e.g.,rheumatoid arthritis and osteoarthritis), neurologic diseases (e.g.,Alzheimer's dementia, amyotrophic lateral sclerosis and Parkinson'sdisease), inflammatory diseases (e.g., sepsis, adult respiratorydistress syndrome, inflammatory bowel disease, and diseminatedintravascular coagulation), fibromyalgia, pain resulting frominflammation (e.g., hyperalgesia and allodynia), neuropathic pain, andcancer (e.g., colon cancer and rectal cancer).

Each of the methods of the invention comprises administering to apatient in need of such treatment or prevention a therapeutically orprophylactically effective amount of norfluoxetine, or apharmaceutically acceptable salt, solvate, hydrate, or clathratethereof. Preferred methods comprise the administration of anenantiomerically pure enantiomer of norfluoxetine, or a pharmaceuticallyacceptable salt, solvate, hydrate, or clathrate thereof. In particularlypreferred methods, the enantiomer of norfluoxetine is R-norfluoxetine.

The invention further encompasses pharmaceutical compositions andpharmaceutical unit dosage forms that comprise norfluoxetine, or apharmaceutically acceptable salt, solvate, hydrate, or clathratethereof. Preferred pharmaceutical compositions and pharmaceutical unitdosage forms comprise an enantiomerically pure enantiomer ofnorfluoxetine (e.g., R-norfluoxetine), or a pharmaceutically acceptablesalt, solvate, hydrate, or clathrate thereof.

3.1. BRIEF DESCRIPTION OF THE DRAWINGS

The unexpected activity of racemic norfluoxetine can be understood withreference to the figures described below:

FIG. 1 shows the inhibition of PGE₂ secretion in cultures of the humanlung fibroblast cell line WI-38 to which 10 μM of various compounds havebeen added, wherein “Dex” is dexamethasone, “Indo” is indomethacin,“NS398” and “celebrex” are specific inhibitors of prostaglandinsynthetase 2, “RS” is racemic fluoxetine, “R” is R-fluoxetine, “S isS-fluoxetine, “nor” is racemic norfluoxetine, and “ami” isamitriptylene.

FIG. 2 shows the inhibition of PGE₂ secretion in cultures of the humanlung fibroblast cell line WI-38 to which 0.5 μM of various compoundshave been added, wherein “Dex” is dexamethasone, “Indo” is indomethacin,“NS398” and “celebrex” are specific inhibitors of prostaglandinsynthetase 2, “RS” is racemic fluoxetine, “R” is R-fluoxetine, “S isS-fluoxetine, “nor” is racemic norfluoxetine, and “ami” isamitriptylene.

3.2. DEFINITIONS

As used herein, the term “prodrug” means a derivative of a compound thatcan hydrolyze, oxidize, or otherwise react under biological conditions(in vitro or in vivo) to provide the compound. Examples of prodrugsinclude, but are not limited to, derivatives of topoisomerase inhibitorsor thalidomide that comprise biohydrolyzable moieties such asbiohydrolyzable amides, biohydrolyzable esters, biohydrolyzablecarbamates, biohydrolyzable carbonates, and biohydrolyzable ureides.

As used herein, the terms “biohydrolyzable carbamate,” “biohydrolyzablecarbonate,” and “biohydrolyzable ureide” mean a carbamate, carbonate, orureide, respectively, of a compound that either: 1) does not interferewith the biological activity of the compound but can confer upon thatcompound advantageous properties in vivo, such as uptake, duration ofaction, or onset of action; or 2) is biologically inactive but isconverted in vivo to the biologically active compound. Examples ofbiohydrolyzable carbamates include, but are not limited to, loweralkylamines, substituted ethylenediamines, aminoacids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines.

As used herein, the term “biohydrolyzable ester” means an ester of acompound that either: 1) does not interfere with the biological activityof the compound but can confer upon that compound advantageousproperties in vivo, such as uptake, duration of action, or onset ofaction; or 2) is biologically inactive but is converted in vivo to thebiologically active compound. Examples of biohydrolyzable estersinclude, but are not limited to, lower alkyl esters, alkoxyacyloxyesters, alkyl acylamino alkyl esters, and choline esters.

As used herein, the term “biohydrolyzable amide” means an amide of acompound that either: 1) does not interfere with the biological activityof the compound but can confer upon that compound advantageousproperties in vivo, such as uptake, duration of action, or onset ofaction; or 2) is biologically inactive but is converted in vivo to thebiologically active compound. Examples of biohydrolyzable amidesinclude, but are not limited to, lower alkyl amides, α-amino acidamides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt prepared from a pharmaceutically acceptable non-toxic inorganic ororganic acid. Suitable non-toxic acids include, but are not limited to,acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric,isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic,nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric,and p-toluenesulfonic acids. For example, specific pharmaceuticallyacceptable salts are hydrochloride and maleic acid salts.

As used herein, the terms “enantiomerically pure,” “pure enantiomer,”and “enantiomerically pure enantiomer” mean a composition that comprisesone enantiomer of a compound and is substantially free of the oppositeenantiomer of the compound. A typical enantiomerically pure compoundcomprises greater than about 80% by weight of one enantiomer of thecompound and less than about 20% by weight of the opposite enantiomer ofthe compound, more preferably greater than about 90% by weight of oneenantiomer of the compound and less than about 10% by weight of theopposite enantiomer of the compound, even more preferably greater thanabout 95% by weight of one enantiomer of the compound and less thanabout 5% by weight of the opposite enantiomer of the compound, and mostpreferably greater than about 99% by weight of one enantiomer of thecompound and less than about 1% by weight of the opposite enantiomer ofthe compound. For example, enantiomerically pure R-norfluoxetinecomprises at least about 80% by weight R-norfluoxetine and less thanabout 20% by weight S-norfluoxetine.

As used herein, a composition that is “substantially free” of a compoundmeans that the composition contains less than about 20% by weight, morepreferably less than about 10% by weight, even more preferably less thanabout 5% by weight, and most preferably less than about 1% by weight ofthe compound. For example, the phrase “R-norfluoxetine substantiallyfree of S-norfluoxetine” is accorded the same meaning as“enantiomerically pure R-norfluoxetine.”

4. DETAILED DESCRIPTION OF THE INVENTION

This invention is based, in part, on the discovery that specificmetabolites of certain serotonin reuptake inhibitors, andenantiomerically pure isomers thereof, exhibit anti-inflammatoryactivity.

A first embodiment of the invention encompasses a method of inhibitingor reducing the production of PGE₂, which comprises contacting a cellthat produces PGE₂ with an amount of norfluoxetine, or apharmaceutically acceptable salt, solvate, hydrate, or clathratethereof, sufficient to inhibit or reduce the production of PGE₂.Preferably, the norfluoxetine is enantiomerically pure R— orS-norfluoxetine. More preferably, the norfluoxetine is enantiomericallypure R-norfluoxetine.

A second embodiment of the invention encompasses a method of treating orpreventing inflammation in a patient (e.g., a mammal, preferably ahuman) which comprises administering to a patient in need of suchtreatment or prevention a therapeutically or prophylactically effectiveamount of norfluoxetine, or a pharmaceutically acceptable salt, solvate,hydrate, or clathrate thereof. Preferably, the norfluoxetine isenantiomerically pure R— or S-norfluoxetine. More preferably, thenorfluoxetine is enantiomerically pure R-norfluoxetine.

A third embodiment of the invention encompasses a method of treating orpreventing a disease or disorder selected from the group consisting ofautoimmune diseases (e.g., lupus erythematosus and multiple sclerosis),arthritis (e.g., rheumatoid arthritis and osteoarthritis), neurologicdiseases (e.g., Alzheimer's dementia, amyotrophic lateral sclerosis andParkinson's disease), inflammatory diseases (e.g., sepsis, adultrespiratory distress syndrome, inflammatory bowel disease, anddiseminated intravascular coagulation), fibromyalgia, pain resultingfrom inflammation (e.g., hyperalgesia and allodynia), neuropathic pain,and cancer (e.g., colon cancer and rectal cancer), which comprisesadministering to a patient in need of such treatment or prevention atherapeutically or prophylactically effective amount of norfluoxetine,or a pharmaceutically acceptable salt, solvate, hydrate, or clathratethereof. Preferably, the norfluoxetine is enantiomerically pure R— orS-norfluoxetine. More preferably, the norfluoxetine is enantiomericallypure R-norfluoxetine.

In each method of treating or preventing a disease or disorder, theracemic or enantiomerically pure norfluoxetine, or pharmaceuticallyacceptable salt, solvate, hydrate, or clathrate thereof, can beadministered in combination with a second active ingredient. Examples ofsecond active ingredients include, but are not limited to,anti-inflammatory drugs, analgesics, proton-pump inhibitors, anduricosuric agents. Examples of anti-inflammatory drugs include, but arenot limited to, corticosteroids, NSAIDs, leukotriene inhibitors, goldcomplexes, and antihistamines.

A fourth embodiment of the invention encompasses a pharmaceuticalcomposition adapted for the treatment or prevention of inflammationwhich comprises norfluoxetine, or a pharmaceutically acceptable salt,solvate, hydrate, or clathrate thereof in an amount sufficient toalleviate or prevent inflammation in a patient. A typical pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient ordiluent. Preferably, the norfluoxetine is enantiomerically pure R— orS-norfluoxetine. More preferably, the norfluoxetine is enantiomericallypure R-norfluoxetine.

A fifth embodiment of the invention encompasses a pharmaceutical unitdosage form adapted for the treatment or prevention of inflammationwhich comprises norfluoxetine, or a pharmaceutically acceptable salt,solvate, hydrate, or clathrate thereof in an amount sufficient toalleviate or prevent inflammation in a patient. A typical pharmaceuticalcomposition further comprises a pharmaceutically acceptable excipient ordiluent. Preferably, the norfluoxetine is enantiomerically pure R— orS-norfluoxetine. More preferably, the norfluoxetine is enantiomericallypure R-norfluoxetine.

Preferred pharmaceutical compositions and unit dosage forms of theinvention comprise a pharmaceutically acceptable excipient or diluent.Specific pharmaceutical compositions and unit dosage forms furthercomprise a second active ingredient. Examples of second activeingredients include, but are not limited to, anti-inflammatory drugs,analgesics, proton-pump inhibitors, and uricosuric agents. Examples ofanti-inflammatory drugs include, but are not limited to,corticosteroids, NSAIDs, leukotriene inhibitors, gold complexes, andantihistamines.

Pharmaceutical unit dosage forms of this invention are suitable fororal, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal),parenteral (e.g., subcutaneous, intravenous, bolus injection,intramuscular, or intraarterial), or transdermal administration to apatient.

A sixth embodiment of the invention encompasses a kit for use in thetreatment or prevention of a disease or disorder which comprises atherapeutically effective amount of norfluoxetine, or a pharmaceuticallyacceptable salt, solvate, hydrate, or clathrate thereof, and atherapeutically effective amount of a second active ingredient.Preferably, the norfluoxetine is enantiomerically pure R— orS-norfluoxetine. More preferably, the norfluoxetine is enantiomericallypure R-norfluoxetine. Examples of second active ingredients include, butare not limited to, anti-inflammatory drugs, analgesics, proton-pumpinhibitors, and uricosuric agents. Examples of anti-inflammatory drugsinclude, but are not limited to, corticosteroids, NSAIDs, leukotrieneinhibitors, gold complexes, and antihistamines.

A seventh embodiment of the invention encompasses a method of preparingan enantiomerically pure enantiomer of norfluoxetine which comprisesasymmetrically reducing an alkyl ester of 3-benzoylpropionic acid,preferably using borane in the presence of a chiral catalyst such as,but not limited to, (S)— or (R)-Me CBS or (+)- or with(−)-chlorodiisopinocamphenylborane (DIPCl®); reacting either theresulting hydroxy ester or lactone with a base in alcohol, preferably,ammonium hydroxide in methanol to (R)— or (S)-4-hydroxy-4-phenylbutyramide; treating with an oxidant such as, but not limited to,iodobenzene diacetate, alkaline bromine, lead tetraacetate, ammoniumtribromide complexes, alkaline solution of N-bromosuccinamide to providea cyclic carbamate; reacting the cyclic carbamate with a base, such aspotassium hydroxide or sodium hydroxide in a solvent such as, but notlimited to, water or isopropanol; and treating with a base and4-chloro-trifluoromethylbenzene to afford enantiomerically pure (R)— or(S)-norfluoxetine.

An eighth embodiment of the invention encompasses novel salts ofenantiomerically pure enantiomers of norfluoxetine and theirpreparations. One such salt is (R)-norfluoxetine•(D)-tartarate. Anotheris (S)-norfluoxetine•(L)-tartarate. The enantiomerically pure salts ofnorfluoxetine can be prepared by contacting (R)— or (S)-norfluoxetine,with a salt such as, but not limited to, (L)- or (D)-tartaric acid and asolvent, preferably methanol.

4.1. Preparation of Norfluoxetine and its Enantiomers and Determinationof Their Biological Activity

Norfluoxetine can be readily prepared using techniques known to thoseskilled in the art. For example, it can be prepared by demethylatingfluoxetine, which in turn can be prepared by methods such as thosedisclosed in U.S. Pat. No. 5,648,396, which is incorporated herein byreference. Demethylation can be achieved using any number ofconventional methods. See, e.g., J. March, Advanced Organic Chemistry407-408 (4^(th) ed.; 1992).

Enantiomerically pure enantiomers of norfluoxetine can be isolated bytechniques known in the art. Examples include, but are not limited to,the formation of chiral salts and the use of chiral or high performanceliquid chromatography (HPLC) and the formation and crystallization ofchiral salts. See, e.g., Jacques, J., et al., Enantiomers, Racemates andResolutions (Wiley-Interscience, New York, 1981); Wilen, S. H., et al.,Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of CarbonCompounds (McGraw-Hill, NY, 1962); and Wilen, S. H., Tables of ResolvingAgents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of NotreDame Press, Notre Dame, Ind., 1972).

A preferred method of preparing enantiomerically pure enantiomers ofnorfluoxetine comprises the asymmetric synthesis of the compound from3-benzoylpropionic acid. According to this method, 3-benzoylpropionicacid is methylated to provide methyl 3-benzylpropionate, which is thencontacted with (+)-B-chlorodiisopinocamphenylborane chloride((+)-DIP—Cl) to provide (R)-χ-phenyl-χ-butrolactone if R-norfluoxetineis being prepared, or with (−)-DIP—Cl to provide(S)-χ-phenyl-χ-butrolactone if S-norfluoxetine is being prepared. Thelactone is subsequently contacted with ammonia to provide thecorresponding chiral 4-hydroxy-4-phenylbutanamide (i.e., (R)— or(S)-4-hydroxy-4-phenylbutanamide). A cyclic carbamate is then preparedfrom the amide by contacting the chiral 4-hydroxy-4-phenylbutanamidewith iodobenzene diacetate. The carbamate is then contacted with base toprovide chiral 3-amino-(1R)-phenyl-propanol-1-ol, which is subsequentlyconverted to the corresponding enantiomer of norfluoxetine free base bycontacting it with sodium hydride and 4-chlorobenzotrifluoride. Thisfree base can then be converted into a salt by known methods. Forexample, (D)-tartaric acid can be contacted with (R)-norfluoxetine freebase to provide (R)-norfluoxetine-(D)-tartarate. Similarly, (L)-tartaricacid can be contacted with (S)-norfluoxetine free base to provide(S)-norfluoxetine-(L)-tartarate.

An example of this method is shown below in Scheme I:

The biological activity of norfluoxetine and enantiomerically pureenantiomers of norfluoxetine can be determined using in vitro and invivo assays known to those skilled in the art. Examples of in vitroassays include, but are not limited to, the use of WI-38 cells. See,e.g., Lin et al., JBC 267:23451-23454 (1992); Patil et al., Blood85:80-86 (1995). In vitro assays using IMR-90 cells have also beenreported. See, e.g., Endo et al., Am. J. Respir. Cell. Md. Biol.12:358-365 (1995). Further examples are provided herein.

4.2. Methods of Treatment and Prevention

This invention encompasses methods of treating and preventing diseaseand disorders which include, but are not limited to, inflammation,autoimmune diseases (e.g., lupus erythematosus and multiple sclerosis),arthritis (e.g., rheumatoid arthritis and osteoarthritis), neurologicdiseases (e.g., Alzheimer's dementia, amyotrophic lateral sclerosis andParkinson's disease), inflammatory diseases (e.g., sepsis, adultrespiratory distress syndrome, inflammatory bowel disease, anddiseminated intravascular coagulation), fibromyalgia, pain resultingfrom inflammation (e.g., hyperalgesia and allodynia), neuropathic pain,and cancer (e.g., colon cancer and rectal cancer) in mammals, and inhumans in particular.

Methods of the invention may, for example, be used to treat or preventchronic and/or acute diseases and conditions. Methods disclosed hereinmay also be used to treat or prevent local and/or systemic conditions.For example, they may be used to treat or prevent inflammation of a widevariety of tissues and organs such as, but are not limited to, the skin,muscles, connective tissue (e.g., tendons and ligaments), blood vessels,nervous tissue, joints, the gastrointestinal tract (e.g., the stomachand the large and small intestine), the liver, the spleen, the lungs,and the kidneys.

Methods of the invention comprise the administration of atherapeutically or prophylactically effective amount of a first activeingredient (e.g., enantiomerically pure R-norfluoxetine, or apharmaceutically acceptable salt, solvate, hydrate, or clathratethereof), optionally in combination with a second active ingredient.Optional second active ingredients can be administered to a patientbefore, concurrently with, or after the administration of the firstactive ingredient by the same or by a different route of administration.

Optional second active ingredients include, but are not limited to,anti-inflammatory drugs, analgesics, proton-pump inhibitors, anduricosuric agents. Examples of anti-inflammatory drugs include, but arenot limited to, corticosteroids, NSAIDs, leukotriene inhibitors, goldcomplexes, and antihistamines.

Examples of corticosteroids include, but are not limited to,alclometasone, amcinonide, beclomethasone, betamethasone, clobetasol,clocortolone, cortisol, prednisolone, and pharmaceutically acceptablesalts, solvates, clathrates, prodrugs, and active metabolites andstereoisomers thereof.

Examples of NSAIDS include, but are not limited to, acetominaphen,apazone, diclofenac, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketorolac, ketoprofen, meclofenamate, mefenamic acid,meloxicam, nabumetone, naproxen, nimesulide, oxaprozin,oxyphenthatrazone, phenylbutazone, piroxicam, salicylates, sulindac,tenoxicam, tolmetin, and pharmaceutically acceptable salts, solvates,clathrates, prodrugs, and active metabolites and stereoisomers thereof.Examples of salicylates include, but are not limited to, aspirin, sodiumsalicylate, choline magnesium trisalicylate, salsalate, diflunisal,salicylsalicylic acid, sulfasalazine, and olsalazine.

Examples of leukotriene inhibitors include, but are not limited to,docebenone, ICI-D2318, MK-0591, MK-886, piripost, zileuton, andpharmaceutically acceptable salts, solvates, clathrates, prodrugs, andactive metabolites and stereoisomers thereof.

Examples of antihistamines include, but are not limited to, acrivastine,astemizole, brompheniramine, carbinoxamine, cetirazine, chlorcyclizine,chlorpheniramine, clemastine, cyclizine, descarboxyloratadine,dimenhydrinate, diphenhydramine, hydroxyzine, levocabastine, loratadine,promethazine, pyrilamine, terfenadine, tripelennamine, andpharmaceutically acceptable salts, solvates, clathrates, prodrugs, andactive metabolites and stereoisomers thereof.

Examples of analgesics include, but are not limited to, bremazocine,buprenorphine, butorphanol, codeine, CTOP, [D-Ala², Glu⁴]deltorphin,DAMGO, diprenorphine, DPDPE, DSLET, dynorphin A, dynorphin B,β-endorphin, ethylketocyclazocine, etorphine, fentanyl,β-funaltrexamine, heroin, hydrocodone, hydromorphone, leu-enkephalin,levophanol, levallorphan, meptazinol, met-enkephalin, methadone,morphine, oxycodone, oxymorphone, nalbuphine, nalmefene, nalorphine,naloxonazine, naloxone, naloxone benzoylhydrazone, naltrexone,naltrindole, α-neoendorphin, nor-binaltorphimine, pentazocine,propoxyphene, spiradoline, 50,488, U69,593, and pharmaceuticallyacceptable salts, solvates, clathrates, prodrugs, and active metabolitesand stereoisomers thereof.

Examples of proton-pump inhibitors include, but are not limited to,prazole derivatives, such as omeprazole, lansoprazole, pantoprazole,rabeprazole, and pharmaceutically acceptable salts, solvates,clathrates, prodrugs, and active metabolites and stereoisomers thereof.Active metabolites of proton-pump inhibitors-can also be employed as anoptional second active ingredient. Examples of such metabolites include,but are not limited to, hydroxy-omeprazole, hydroxy-lansoprazole, thecarboxylic acid derivative of omeprazole, and desmethyl-pantoprazole.

Examples of uricosuric agents include, but are not limited to,allopurinol, benzbromarone, colchicine, probenecid, sulfinpyrazone, andpharmaceutically acceptable salts, solvate, hydrates, clathrates, andactive metabolites and stereoisomers thereof.

The magnitude of a prophylactic or therapeutic dose of each activeingredient in the acute or chronic management of a disease or disorderwill vary with the disorder itself, the specific active ingredients, andthe route of administration. The dose and/or dose frequency may alsovary according to age, body weight, response, and the past medicalhistory of the patient. Suitable dosing regimens can be readily selectedby those skilled in the art with due consideration of such factors byfollowing, for example, dosages reported in the literature andrecommended in the Physician's Desk Reference® (54^(th) ed., 2000).

Unless otherwise indicated, the magnitude of a prophylactic ortherapeutic dose of each active ingredient used in an embodiment of theinvention will be that which is safe and effective (e.g., has receivedregulatory approval).

In one embodiment of the invention, a first active ingredient (e.g.,enantiomerically pure R— or S-norfluoxetine) is administered orally asneeded in an amount of from about 1 mg to about 500 mg, preferably in anamount of from about 10 mg to about 300 mg, more preferably in an amountof from about 15 mg to about 200 mg, and most preferably in an amount offrom about 20 mg to about 100 mg. The dosage amounts and frequenciesprovided above are encompassed by the terms “therapeutically effective,”“prophylactically effective,” and “therapeutically or prophylacticallyeffective” as used herein.

The suitability of a particular route of administration employed for aparticular active ingredient will depend on the active ingredient itself(e.g., whether it can be administered orally without decomposing priorto entering the blood stream) and the disease or disorder to be treatedor prevented. For example, topical administration is typically preferredfor treating or preventing local inflammation of the skin, while oral orparenteral administration is typically preferred for systemicinflammation or inflammation within the body. Similarly, oral orparenteral administration may be preferred for the treatment orprevention of acute inflammation, whereas transdermal or subcutaneousroutes of administration may be employed for treatment or prevention ofa chronic disease or disorder.

The optional administration of a second active ingredient will alsodepend on the disease or disorder to be treated or prevented. Forexample, uricosuric agents are preferably used in the treatment orprevention of inflammation of the bowel (e.g., gerd), while goldcomplexes are preferably used in the treatment or prevention ofinflammation of the joints (e.g., rheumatoid arthritis).

4.3. Pharmaceutical Compositions and Dosage Forms

Pharmaceutical compositions and dosage forms of the invention compriseone or more of the active ingredients disclosed herein (e.g.,enantiomerically pure R— or S-norfluoxetine, or a pharmaceuticallyacceptable prodrug, salt, solvate, hydrate, or clathrate thereof).Pharmaceutical compositions and dosage forms of the invention typicallyalso comprise one or more pharmaceutically acceptable excipients ordiluents.

Single unit dosage forms of the invention are suitable for oral, mucosal(e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g.,subcutaneous, intravenous, bolus injection, intramuscular, orintraarterial), or transdermal administration to a patient. Examples ofdosage forms include, but are not limited to: tablets; caplets;capsules, such as soft elastic gelatin capsules; cachets; troches;lozenges; dispersions; suppositories; ointments; cataplasms (poultices);pastes; powders; dressings; creams; plasters; solutions; patches;aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage formssuitable for oral or mucosal administration to a patient, includingsuspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The composition, shape, and type of dosage forms of the invention willtypically vary depending on their use. For example, a dosage form usedin the acute treatment of inflammation or a related disorder may containlarger amounts of one or more of the active ingredients it comprisesthan a dosage form used in the chronic treatment of the same disease.Similarly, a parenteral dosage form may contain smaller amounts of oneor more of the active ingredients it comprises than an oral dosage formused to treat the same disease or disorder. These and other ways inwhich specific dosage forms encompassed by this invention will vary fromone another will be readily apparent to those skilled in the art. See,e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing,Easton Pa. (1990).

Typical pharmaceutical compositions and dosage forms comprise one ormore excipients. Suitable excipients are well known to those skilled inthe art of pharmacy, and non-limiting examples of suitable excipientsare provided herein. Whether a particular excipient is suitable forincorporation into a pharmaceutical composition or dosage form dependson a variety of factors well known in the art including, but not limitedto, the way in which the dosage form will be administered to a patient.For example, oral dosage forms such as tablets may contain excipientsnot suited for use in parenteral dosage forms. The suitability of aparticular excipient may also depend on the specific active ingredientsin the dosage form. For example, the decomposition of some activeingredients, and norfluoxetine and its enantiomerically pure enantiomersin particular, can be accelerated by some excipients such as lactose, orwhen exposed to water. Active ingredients that comprise primary orsecondary amines (e.g., norfluoxetine and its enantiomerically pureenantiomers) are particularly susceptible to such accelerateddecomposition. Consequently, this invention encompasses pharmaceuticalcompositions and dosage forms that contain little, if any, lactose othermono- or di-saccharides. As used herein, the term “lactose-free” meansthat the amount of lactose present, if any, is insufficient tosubstantially increase the degradation rate of an active ingredient.

Lactose-free compositions of the invention can comprise excipients thatare well known in the art and are listed, for example, in the U.S.Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose-freecompositions comprise active ingredients, a binder/filler, and alubricant in pharmaceutically compatible and pharmaceutically acceptableamounts. Preferred lactose-free dosage forms comprise activeingredients, microcrystalline cellulose, pre-gelatinized starch, andmagnesium stearate.

This invention further encompasses anhydrous pharmaceutical compositionsand dosage forms comprising active ingredients, since water canfacilitate the degradation of some compounds. For example, the additionof water (e.g., 5%) is widely accepted in the pharmaceutical arts as ameans of simulating long-term storage in order to determinecharacteristics such as shelf-life or the stability of formulations overtime. See, e.g., Jens T. Carstensen, Drug Stability: Principles &Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect,water and heat accelerate the decomposition of some compounds. Thus, theeffect of water on a formulation can be of great significance sincemoisture and/or humidity are commonly encountered during manufacture,handling, packaging, storage, shipment, and use of formulations.

Anhydrous pharmaceutical compositions and dosage forms of the inventioncan be prepared using anhydrous or low moisture containing ingredientsand low moisture or low humidity conditions. Pharmaceutical compositionsand dosage forms that comprise lactose and at least one activeingredient that comprises a primary or secondary amine are preferablyanhydrous if substantial contact with moisture and/or humidity duringmanufacturing, packaging, and/or storage is expected.

An anhydrous pharmaceutical composition should be prepared and storedsuch that its anhydrous nature is maintained. Accordingly, anhydrouscompositions are preferably packaged using materials known to preventexposure to water such that they can be included in suitable formularykits. Examples of suitable packaging include, but are not limited to,hermetically sealed foils, plastics, unit dose containers (e.g., vials),blister packs, and strip packs.

The invention further encompasses pharmaceutical compositions and dosageforms that comprise one or more compounds that reduce the rate by whichan active ingredient will decompose. Such compounds, which are referredto herein as “stabilizers,” include, but-are not limited to,antioxidants such as ascorbic acid, pH buffers, or salt buffers.

Like the amounts and types of excipients, the amounts and specific typesof active ingredients in a dosage form may differ depending on factorssuch as, but not limited to, the route by which it is to be administeredto patients. However, typical dosage forms of the invention compriseracemic or enantiomerically pure norfluoxetine, or a pharmaceuticallyacceptable salt, solvate, clathrate, hydrate, or prodrug thereof in anamount of from about 1 mg to about 500 mg, preferably in an amount offrom about 10 mg to about 300 mg, more preferably in an amount of fromabout 15 mg to about 200 mg, and most preferably in an amount of fromabout 20 mg to about 100 mg.

4.3.1. Oral Dosage Forms

Pharmaceutical compositions of the invention that are suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms of the invention are prepared by combining theactive ingredient(s) in an intimate admixture with at least oneexcipient according to conventional pharmaceutical compoundingtechniques. Excipients can take a wide variety of forms depending on theform of preparation desired for administration. For example, excipientssuitable for use in oral liquid or aerosol dosage forms include, but arenot limited to, water, glycols, oils, alcohols, flavoring agents,preservatives, and coloring agents. Examples of excipients suitable foruse in solid oral dosage forms (e.g., powders, tablets, capsules, andcaplets) include, but are not limited to, starches, sugars,micro-crystalline cellulose, diluents, granulating agents, lubricants,binders, and disintegrating agents.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms, in which case solidexcipients are employed. If desired, tablets can be coated by standardaqueous or nonaqueous techniques. Such dosage forms can be prepared byany of the methods of pharmacy. In general, pharmaceutical compositionsand dosage forms are prepared by uniformly and intimately admixing theactive ingredients with liquid carriers, finely divided solid carriers,or both, and then shaping the product into the desired presentation ifnecessary.

For example, a tablet can be prepared by compression or molding.Compressed tablets can be prepared by compressing in a suitable machinethe active ingredients in a free-flowing form such as powder orgranules, optionally mixed with an excipient. Molded tablets can be madeby molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

Examples of excipients that can be used in oral dosage forms of theinvention include, but are not limited to, binders, fillers,disintegrants, and lubricants. Binders suitable for use inpharmaceutical compositions and dosage forms include, but are notlimited to, corn starch, potato starch, or other starches, gelatin,natural and synthetic gums such as acacia, sodium alginate, alginicacid, other alginates, powdered tragacanth, guar gum, cellulose and itsderivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethylcellulose calcium, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose; pre-gelatinized starch, hydroxypropylmethyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystallinecellulose, and mixtures thereof.

Suitable forms of microcrystalline cellulose include, but are notlimited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICELRC-581, AVICEL-PH-105 (available from FMC Corporation, American ViscoseDivision, Avicel Sales, Marcus Hook, PA), and mixtures thereof. Anspecific binder is a mixture of microcrystalline cellulose and sodiumcarboxymethyl cellulose sold as AVICEL RC-58 1. Suitable anhydrous orlow moisture excipients or additives include AVICEL-PH-103™ and Starch1500 LM.

Examples of fillers suitable for use in the pharmaceutical compositionsand dosage forms disclosed herein include, but are not limited to, talc,calcium carbonate (e.g., granules or powder), microcrystallinecellulose, powdered cellulose, dextrates, kaolin, mannitol, silicicacid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.The binder or filler in pharmaceutical compositions of the invention istypically present in from about 50 to about 99 weight percent of thepharmaceutical composition or dosage form.

Disintegrants are used in the compositions of the invention to providetablets that disintegrate when exposed to an aqueous environment.Tablets that contain too much disintegrant may disintegrate in storage,while those that contain too little may not disintegrate at a desiredrate or under the desired conditions. Thus, a sufficient amount ofdisintegrant that is neither too much nor too little to detrimentallyalter the release of the active ingredients should be used to form solidoral dosage forms of the invention. The amount of disintegrant usedvaries based upon the type of formulation, and is readily discernible tothose of ordinary skill in the art. Typical pharmaceutical compositionscomprise from about 0.5 to about 15 weight percent of disintegrant,preferably from about 1 to about 5 weight percent of disintegrant.

Disintegrants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate, potato or tapioca starch, other starches, pre-gelatinizedstarch, other starches, clays, other algins, other celluloses, gums, andmixtures thereof.

Lubricants that can be used in pharmaceutical compositions and dosageforms of the invention include, but are not limited to, calciumstearate, magnesium stearate, mineral oil, light mineral oil, glycerin,sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid,sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanutoil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, andsoybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, andmixtures thereof. Additional lubricants include, for example, a syloidsilica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore,Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co.of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold byCabot Co. of Boston, Mass.), and mixtures thereof If used at all,lubricants are typically used in an amount of less than about 1 weightpercent of the pharmaceutical compositions or dosage forms into whichthey are incorporated.

An example of a preferred liquid oral dosage form of the inventioncomprises 20 mg/5 mL R-norfluoxetine hydrochloride in a solutioncontaining alcohol 0.23%, benzoic acid, flavoring agent, glycerin,purified water, and sucrose. A preferred caplet dosage form of theinvention comprises 20 mg R-norfluoxetine hydrochloride, gelatin, ironoxide, silicone, starch, and titanium dioxide.

4.3.2. Delayed Release Dosage Forms

Active ingredients of the invention can be administered by controlledrelease means or by delivery devices that are well known to those ofordinary skill in the art. Examples include, but are not limited to,those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809;3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548,5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which isincorporated herein by reference. Such dosage forms can be used toprovide slow or controlled-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Suitablecontrolled-release formulations known to those of ordinary skill in theart, including those described herein, can be readily selected for usewith the active ingredients of the invention. The invention thusencompasses single unit dosage forms suitable for oral administrationsuch as, but not limited to, tablets, capsules, gelcaps, and capletsthat are adapted for controlled-release.

All controlled-release pharmaceutical products have a common goal ofimproving drug therapy over that achieved by their non-controlledcounterparts. Ideally, the use of an optimally designedcontrolled-release preparation in medical treatment is characterized bya minimum of drug substance being employed to cure or control thecondition in a minimum amount of time. Advantages of controlled-releaseformulations include extended activity of the drug, reduced dosagefrequency, and increased patient compliance. In addition,controlled-release formulations can be used to affect the time of onsetof action or other characteristics, such as blood levels of the drug,and can thus affect the occurrence of side (e.g., adverse) effects.

Most controlled-release formulations are designed to initially releasean amount of drug (active ingredient) that promptly produces the desiredtherapeutic effect, and gradually and continually release of otheramounts of drug to maintain this level of therapeutic or prophylacticeffect over an extended period of time. In order to maintain thisconstant level of drug in the body, the drug must be released from thedosage form at a rate that will replace the amount of drug beingmetabolized and excreted from the body. Controlled-release of an activeingredient can be stimulated by various conditions including, but notlimited to, pH, temperature, enzymes, water, or other physiologicalconditions or compounds.

4.3.3. Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including, but not limited to, subcutaneous, intravenous(including bolus injection), intramuscular, and intraarterial. Becausetheir administration typically bypasses patients' natural defensesagainst contaminants, parenteral dosage forms are preferably sterile orcapable of being sterilized prior to administration to a patient.Examples of parenteral dosage forms include, but are not limited to,solutions ready for injection, dry products ready to be dissolved orsuspended in a pharmaceutically acceptable vehicle for injection,suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude, but are not limited to: Water for Injection USP; aqueousvehicles such as, but not limited to, Sodium Chloride Injection,Ringer's Injection, Dextrose Injection, Dextrose and Sodium ChlorideInjection, and Lactated Ringer's Injection; water-miscible vehicles suchas, but not limited to, ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as, but not limitedto, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Compounds that increase the solubility of one or more of the activeingredients disclosed herein can also be incorporated into theparenteral dosage forms of the invention.

4.3.4. Transdermal, Tropical, and Mucosal Dosage Forms

Transdermal, topical, and mucosal dosage forms of the invention include,but are not limited to, ophthalmic solutions, sprays, aerosols, creams,lotions, ointments, gels, solutions, emulsions, suspensions, or otherforms known to one of skill in the art. See, e.g., Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed.,Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treatingmucosal tissues within the oral cavity can be formulated as mouthwashesor as oral gels. Further, transdermal dosage forms include “reservoirtype” or “matrix type” patches, which can be applied to the skin andworn for a specific period of time to permit the penetration of adesired amount of active ingredients.

Suitable excipients (e.g., carriers and diluents) and other materialsthat can be used to provide transdermal, topical, and mucosal dosageforms encompassed by this invention are well known to those skilled inthe pharmaceutical arts, and depend on the particular tissue to which agiven pharmaceutical composition or dosage form will be applied. Withthat fact in mind, typical excipients include, but are not limited to,water, acetone, ethanol, ethylene glycol, propylene glycol,butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil,and mixtures thereof to form lotions, tinctures, creams, emulsions, gelsor ointments, which are non-toxic and pharmaceutically acceptable.Moisturizers or humectants can also be added to pharmaceuticalcompositions and dosage forms if desired. Examples of such additionalingredients are well known in the art. See, e.g., Remington'sPharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa.(1980 & 1990).

Depending on the specific tissue to be treated, additional componentsmay be used prior to, in conjunction with, or subsequent to treatmentwith active ingredients of the invention. For example, penetrationenhancers can be used to assist in delivering the active ingredients tothe tissue. Suitable penetration enhancers include, but are not limitedto: acetone; various alcohols such as ethanol, oleyl, andtetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethylacetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such aspolyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; andvarious water-soluble or insoluble sugar esters such as Tween 80(polysorbate 80) and Span 60 (sorbitan monostearate).

The pH of a pharmaceutical composition or dosage form, or of the tissueto which the pharmaceutical composition or dosage form is applied, mayalso be adjusted to improve delivery of one or more active ingredients.Similarly, the polarity of a solvent carrier, its ionic strength, ortonicity can be adjusted to improve delivery. Compounds such asstearates can also be added to pharmaceutical compositions or dosageforms to advantageously alter the hydrophilicity or lipophilicity of oneor more active ingredients so as to improve delivery. In this regard,stearates can serve as a lipid vehicle for the formulation, as anemulsifying agent or surfactant, and as a delivery-enhancing orpenetration-enhancing agent. Different salts, hydrates or solvates ofthe active ingredients can be used to further adjust the properties ofthe resulting composition.

4.3.5. Kits

Typically, active ingredients of the invention are preferably notadministered to a patient at the same time or by the same route ofadministration. This invention therefore encompasses kits which, whenused by the medical practitioner, can simplify the administration ofappropriate amounts of active ingredients to a patient.

A typical kit of the invention comprises a unit dosage form of racemicor enantiomerically pure norfluoxetine, or a pharmaceutically acceptableprodrug, salt, solvate, hydrate, or clathrate thereof, and a unit dosageform of a second active ingredient. Examples of second activeingredients include, but are not limited to, anti-inflammatory drugs,analgesics, and proton-pump inhibitors. Examples of secondanti-inflammatory drugs include, but are not limited to,corticosteroids, NSAIDs, gold complexes, and antihistamines.

Kits of the invention can further comprise devices that are used toadminister the active ingredients. Examples of such devices include, butare not limited to, syringes, drip bags, patches, and inhalers.

Kits of the invention can further comprise pharmaceutically acceptablevehicles that can be used to administer one or more active ingredients.For example, if an active ingredient is provided in a solid form thatmust be reconstituted for parenteral administration, the kit cancomprise a sealed container of a suitable vehicle in which the activeingredient can be dissolved to form a particulate-free sterile solutionthat is suitable for parenteral administration. Examples ofpharmaceutically acceptable vehicles include, but are not limited to:Water for Injection USP; aqueous vehicles such as, but not limited to,Sodium Chloride Injection, Ringer's Injection, Dextrose Injection,Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as, but not limited to, ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as, but not limited to, corn oil, cottonseed oil, peanut oil,sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

5. EXAMPLES

Certain embodiments of the invention, as well as certain novel andunexpected advantages of the invention, are illustrated by the followingnon-limiting examples.

5.1. Example 1 Synthesis of R-Norfluoxetine·-(D)-Tartarate

The 3-benzoylpropionic acid starting material is available from Aldrich,Acros and Lancaster. The methyl ester (Methyl 3-benzoylpropionate) isalso available from Lancaster.

Methyl 3-Benzoylpropionate (1): To a 1.0 L round bottom flask was added89 g (0.5 mol) of 3-benzoylpropionic acid and 200 mL of methanol. Thesolution was stirred until all of the acid had dissolved. To thissolution was added approximately 2.0 mL of conc. H₂SO₄ and the reactionrefluxed for 2.5 hours. After the reaction was determined to becomplete, the methanol was removed on the rotoevaporator and to theresidual oil was added ethyl acetate (˜500 mL). To the ethyl acetatelayer was then added 150 mL of a saturated sodium carbonate solution.The layers were extracted and allowed to separate. The organic layer wasthen dried (MgSO₄), filtered, and concentrated to yield methyl3-benzoylpropionate (1) as a light yellow oil. Yield 96 g; yield100%; >97% cp. Lit bp 172-174° C. at 10 mm Hg.; ¹H NMR ppm (δ), CDCl₃7.98 (dd, 2H), 7.54 (m, 1H), 7.45 (m, 2H), 3.70 (s, 3H, OCH₃), 3.32 (t,2H), 2.76 (t, 2H). ¹³C NMR ppm (δ), CDCl₃ 198.0 (C═O), 173.3 (CO₂),136.5 (q), 133.2, 128.6, 128.0, 51.7 (OCH₃), 33.3, 28.0. IR oil cm⁻¹2952, 1737, 1687, 1596, 1449, 1358, 1221, 1168 1001, 750, 691.

Preparation of (R)-γ-phenyl-γ-butrolactone (2): To a 500 mL round bottomflask was added 30.5 g (0.0951 mol) of (+)-DIP—Cl[(−)-B-chlorodiisopinocamphenylborane]. To this was added 30 mL of dryTHF at room temperature over 15 minutes. Once dissolved, the solutionwas cooled to 0° C. While maintaining the temperature 0° C., to thissolution was added dropwise 10.80 g (0.0561 mol) of methyl3-benzoylpropionate (1) in 15 mL of dry THF over 30 minutes.

During the addition of the ketoester, the reaction mixture turned milkywhite in color and after all of the material was added the reactionmixture turned clear. The reaction was maintained between 0° C. for 8hours, and the reaction progress was monitored by HPLC using the methodoutlined previously. After 8 hours, the reaction mixture was allowed towarm to 22° C. The reaction was stopped after all of the startingmaterial was gone. [typical retention time: Ketoester (IIa)=6.9 minutes;Alcoholester (III)=4.9 min]. To the reaction mixture was then addedslowly 26 mL of water (1.44 mol) over 10 minutes keeping the temperaturebelow 10° C. Methanol was then added (66 mL, 1.63 mol) followed by 76 mLof a 5 M aqueous NaOH solution keeping the temperature below 10° C. Themixture was stirred for 2 hours and checked periodically by HPLC. After2 hours the reaction was shown to be complete [Retention Time AlcoholAcid (IV)=3.4 min]. The light yellow solution was poured into 500 mL ofMTBE and 200 mL of saturated NaHCO₃ solution. After the extraction thelayers were separated and the aqueous layer re-extracted with 200 mL ofMTBE. The layers were again separated and the aqueous layer acidified topH=2 with concentrated HCl. The aqueous layer was saturated with NaCland then extracted 3 times with 300 mL of ethyl acetate. The ethylacetate layer was rotoevaporated and the residue taken up in toluene(1000 mL). To the toluene was added 1.5 g (0.006 mol) of PPTS and heatedto reflux for 2 hours until the lactonization was complete. The solutionwas cooled to room temperature and washed twice with an saturated aq.NaHCO₃ solution. The toluene layer was rotoevaporated to yield 8.36 g(92%) of the lactone (2). The chemical purity is 97.3%, and the opticalpurity is 98.75 ip (shown below). Achiral HPLC Retention Time=5.8 min.Chiral Retention Time=26.0 min. (R), 30.1 min. (S). ¹H NMR ppm (δ),CDCl₃ 7.34-7.25 (m, 5H), 5.47 (t, 1H), 2.68-2.60 (m, 3H), 2.22-2.10 (m,1H). ¹³C NMR ppm (δ), CDCl₃ 176.9 (C═O), 139.3(q), 128.6, 128.3, 125.2,81.1 (C—O), 30.8, 28.8.

Preparation of methyl (R)-4-hydroxy-4-phenylbutyrate (5): To a dry 250mL round bottom flask were added (S)-Me-CBS (5 mL, 1.0 M in toluene, 5.0mmol) and 50 mL of THF under argon. The solution was cooled to −5° C. Tothis solution were added simultaneously BH₃∘THF (50 mL, 10M in THF, 50mmol) and ketoester 1 in neat (9.6 g, 50 mmol) via syringe. The additionwas completed in 30 min. The reaction mixture was stirred at the sametemperature for another 30 minutes and worked up by addition of aqueousK₂CO₃ solution. After stirring for 30 minutes, the organic layer wasseparated, and the aqueous layer was extracted with AcOEt. The combinedorganic layers were washed with brine and dried over (Na₂SO₄). Revomalof solvent gave a pale yellow oil, containing the hydroester 5 and theCBS catalyst (ca. 11 g). The crude product could be used directly fornext reaction. A sample was purified by hydrolysis with 3 N NaOH, andconverted into lactone 2 by azeotropically reflux with toluene. E.e wasmeasured by HPLC on lactone 2 (95.6%)

Preparation of (R)-4-hydroxy-4-phenylbutanamide (6): To a 100 mL roundbottom flask was added 9 g (55.56 mmol) of the chiral lactone (2). Tothis oil was added 30 mL of methanol and 28-30% aqueous ammonia (30 mL,257 mmol). This solution was stirred at room temperature for 6 hourswhich at this point the reaction completed. The ammonia in methanol wasremoved on the rotoevaporator and to the residual oil was addedapproximately 20 mL of ethyl acetate. The ethyl acetate was allowed toevaporate slowly and yielded a white solid (11.12 g, 100%), mp=74-75 °C. ¹H NMR ppm (δ), CDCl₃ 7.35-7.24 (m, 5H), 5.79 (s, 2H, NH₂), 4.77 (dd,1H,CH—O), 3.66 (s, 1H, OH), 2.40-2.21 (m, 2H), 2.11-2.00 (m, 2H). ¹³CNMR ppm (δ), CDCl₃ 176.4 (C═O), 144.6(q), 128.7, 127.7, 126.0, 73.7(C—O), 34.3, 32.3.

Preparation of the Cyclic Carbamate (7U: To a 250 mL round bottom flaskwas added 11 g (62.12 mmol) of the chiral alcohol amide (6). Thematerial was dissolved in 150 mL of acetonitrile and then added with20.8 g (64.60 mmol) of iodobenzene diacetate. The solution was stirredat 40° C. for 5 hours monitoring the reaction by HPLC. After thereaction was complete, the acetonitrile was removed on therotoevaporator. The crude pale yellow solide (9.1 g, 83%) wasrecrystallized from ethyl acetate to give 5.5 g of the pure product (51%yield). Chemical purity=100%, Isomeric Purity=99.7%, mp=191-192° C.Achiral HPLC Retention Time=3.6 min. Chiral Retention Time=18.1 min.(R), 22.7 min. (S). ¹H NMR ppm (δ), CDCl₃ 7.37-7.29 (m, 5H), 6.65 (s,1H, NH), 5.34 (dd, 1H,CH—O), 3.48-3.36 (m, 2H), 2.19-2.05 (m, 2H). ¹³CNMR ppm (δ), CDCl₃ 155.3 (C═O), 139.3 (q), 128.8, 128.6, 125.9, 78.8(C—O), 39.0, 28.9. IR KBr cm⁻¹ 3303, 1690, 1664, 1482, 1457, 20 1300,1137, 1051, 767, 756, 697.

Preparation of 3-amino-(1R)-phenyl-propan-1-ol (8): To a 1 L roundbottom flask was added 66 g (372.88 mmol) of the cyclic carbamate (7).To this was added 200 mL of isopropanol, 200 mL of H₂O followed by 45 gof KOH (803.57 mmol, 2.2 equiv). This solution was refluxed for 3 hoursand then allowed to cool to room temperature. The IPA layer wasseparated and the aqueous layer was extracted with IPA once more (50mL). The combined organic layers were washed with brine (1×15 mL), andconcentrated on the rotoevaporator to obtain an oil. The oil was driedby azeotropically reflux with toluene and used for next reaction. 47 gof 8 was obtained as pale yellow oil (84%). ¹H NMR ppm (δ), CDCl₃7.58-7.20 (m, 5H), 4.95 (dd, 1H), 3.10 (m, 1H), 2.98 (m, 1H), 1.94-1.70(m, 2H). ¹³C NMR ppm (δ), CDCl₃ 145.35, 128.53, 127.28, 125.91, 75.67,40.79, 40.09.

Preparation of Norfluoxetine Free Base (9): To a 100 mL round bottomflask was added 4 g (26.88 mmol) of the aminoalcohol (8) and this wasdissolved in 80 mL of DMSO. To this solution was then added 1.5 g (37.5mmol) of sodium hydride (60 wt % in oil, washed with hexanes) at 0° C.The solution was stirred at ambient temperature for 5 minutes, thenheated to 70° C. for 30 min. To this dark orange solution was added 6.9g of 4-chlorobenzotrifluoride (38.21 mmol). The reaction mixture washeated to 115° C. for 3 hours. The reaction was allowed to cool to roomtemperature and then poured into 15 mL of ice water to quench thereaction. The reaction mixture was extracted with ethyl ether (5×50 mL),followed by two extractions using toluene (2×50 mL). The organic layerswere combined and washed twice with brine. The aqueous layer wasseparated and the organic layer dried, filtered, and rotoevaporated.This crude material was purified by column chromatography (silica gel)using 5% methanol:methylene chloride as the eluent. The yield of purenorfluoxetine free-base (9) is 4.1 g (69%). 1.1 g of the startingmaterial was recovered. ¹H NMR ppm (δ), CDCl₃ 7.57 (d, 2H), 7.44-7.24(m, 5H), 6.91 (d, 2H), 5.43 (dd, 1H), 3.15 (t, 2H), 2.39 (m, 1H), 2.28(m, 1H). ¹³C NMR ppm (δ), CDCl₃ 160.84, 141.34, 129.06, 128.09, 127.04,127.00, 126.03, 116.02, 78.64, 42.54, 38.80.

Preparation of (R)-Norfluoxetine∘(D)-Tartrate (10): To a 50 mL roundbottom flask was added 1.63 g (5.53 mmol) of (R)-fluoxetine free amine(9). To this was added 20 mL of methanol and the amine was dissolved. Tothis solution was added 840 mg of (D)-tartaric acid (5.6 mmol). Thereaction mixture was stirred for 30 minutes and then methanol wasrotoevaporated to dryness. The solid was slurred in 20 mL of TBME andfiltered. The white solid was dried in a vacuum oven for 5 hours at ˜40°C. 2.08 g of (R)-norfluoxetine (D)-tartrate was obtained (85%). 99.44%ee, 99.53 A % purity. ¹H NMR ppm (δ), DMSO 7.63-7.10 (m, 9H), 5.61 (m,1H, CH—O), 3.90 (dd, 2H), 2.90 (m, 2H), 2.50 (s, 1H), 2.35-2.08 (r,2H).¹³C NMR ppm (δ), DMSO 175.3, 160.8, 140.8, 129.5, 128.71, 127.5, 126.6,117.0, 77.0, 72.3, 36.4.

5.2. Example 2 Inhibition of PGE₂ Production

Compounds were tested for anti-inflammatory activity using a wholecell-based assay, wherein the human lung fibroblast cell line WI-38 wascultured in Eagle's basal media supplemented with 10% fetal calf serum(FCS) in 48-well tissue culture dishes in a humidified 7% CO₂ atmosphereat 37° C. When the cells reached confluence, they were fed with freshmedia for 24 hours. The media was removed and fresh media, containingeither 5% or 10% FCS, was added to the cells. Compounds were added in avolume of 1 μL in either ethanol or dimethylsulfoxide (DMSO) to give thedesired final concentration. Interleukin-1β (Calbiochem) in 0.1% bovineserum albumin in phosphate buffered saline was added in a 1 μL aliquotto give a final concentration of 100 pg/mL. The cells were cultured for24 hours and the amount of PGE₂ secreted into the cell culture media wasassayed using a PGE₂ enzyme immunoassay kit (Cayman Chemicals, AnnArbor, Mich.). Inhibition of PGE₂ production is indicative ofanti-inflammatory activity. This assay will detect either directinhibition of the prostaglandin synthetase 2 enzyme as evidenced by theefficacy of celebrex, or it will detect the inhibition oftranscription—or the activation of transcription— of the protaglandinsynthetase 2 gene as evidenced by the efficacy of dexamethasone.

Using this assay, the effect of racemic or enantiomerically purenorfluoxetine in vitro is shown in FIGS. 1 and 2. Additional results areshown below in Tables 1-4. TABLE 1 (10% fetal calf serum-containingmedia) Sample PGE₂ (pg/ml) No IL-1β low +IL-1β 886 celebrex 0.5 uM lowcelebrex 10 uM low rac-fluoxetine 0.5 uM low rac-fluoxetine 10 uM lowrac-norfluoxetine 0.5 uM 189 rac-norfluoxetine 10 uM low(R)-norfluoxetine 0.5 uM 714 (R)-norfluoxetine 10 uM 159(S)-norfluoxetine 0.5 uM 809 (S)-norfluoxetine 10 uM 486 rac-ketoprofin10 uM low (R)-ketoprofin 0.5 uM low (R)-ketoprofin 10 uM low(S)-ketoprofin 0.5 uM low (S)-ketoprofin 10 uM low

TABLE 2 (5% fetal calf serum-containing media) Sample PGE₂ (pg/ml) NoIL-1β low +IL-1β 667 celebrex 0.5 uM  16 celebrex 10 uM lowrac-fluoxetine 0.5 uM low rac-fluoxetine 10 uM low rac-norfluoxetine 0.5uM low rac-norfluoxetine 10 uM low (R)-norfluoxetine 0.5 uM 255(R)-norfluoxetine 10 uM  31 (S)-norfluoxetine 0.5 uM 483(S)-norfluoxetine 10 uM 112 rac-ketoprofen 0.5 uM 119 rac-ketoprofen 10uM low (R)-ketoprofen 0.5 uM low (R)-ketoprofen 10 uM low (S)-ketoprofen0.5 uM 145 (S)-ketoprofen 10 uM low

TABLE 3 (10% fetal calf serum-containing media) Sample PGE₂ (pg/ml) NoIL-1β low +IL-1β 1980 celebrex 0.5 uM low celetrex 10 uM lowrac-fluoxetine 0.5 uM 545 rac-fluoxetine 10 uM 156 nortriptylene 0.5 uM2040 nortriptylene 10 uM 648 No IL-1β low No IL-1β low No IL-1β low NoIL-1β 8 +IL-1β 5800 +IL-1β 2760 +IL-1β 5630 +IL-1β 10970

TABLE 4 (5% fetal calf serum-containing media) Sample PGE₂ (pg/ml) NoIL-1β low +IL-1β 909 celebrex 0.5 uM low celebrex 10 uM lowrac-fluoxetine 0.5 uM 22 rac-fluoxetine 10 uM low nortriptylene 0.5 uM880 nortriptylene 10 uM 331 No IL-1β low No IL-1β low No IL-1β low NoIL-1β low +IL-1β 1520 +IL-1β 1250 +IL-1β 1500 +IL-1β 3870

Surprisingly, these results indicate that enantiomerically pure R— andS-norfluoxetine exhibit substantially equipotent anti-inflammatoryactivity in vitro, whereas S-norfluoxetine is known to be approximately20-fold more active than R-norfluoxetine in the inhibition of serotoninreuptake.

5.3. Example 3 Determination of IC₅₀ Values

The IC₅₀ values of enantiomerically pure R— and S-norfluoxetine weredetermined by incubation of the WI-38 cells with varying concentrationsof the compounds in question (0.009, 0.019, 0.039, 0.078, 0.156, 0.3125,0.625, 1.25, 2.5, 5, 10, and 20 μm). The PGE₂ produced for eachconcentration was plotted against the concentration of compound usingthe 6 safit 4.0 program (Erithacus software) and the IC₅₀ determined byfitting the data to the 4-parameter equation. From these tests, it wasdetermined that the IC₅₀ of R-norfluoxetine is about 0.79 uM, and thatof S-norfluoxetine is about 1.4 uM.

It is noteworthy that these values, especially that of R-norfluoxetine,fall within values that may be obtained in serum in humans. For example,the package insert for Prozac® states that serum levels of norfluoxetinerange from 72 to 258 ng/ml (approximately 0.23 to 0.83 uM) aftertreatment for 30 days with 40 mg per day of fluoxetine.

While the invention has been described with respect to the particularembodiments, it will be apparent to those skilled in the art thatvarious changes and modifications may be made without departing from thespirit and scope of the invention as defined in the claims. Suchmodifications are also intended to fall within the scope of the appendedclaims.

1-17. (canceled)
 18. A pharmaceutical composition comprisingnorfluoxetine, or a pharmaceutically acceptable salt, solvate, hydrate,or clathrate thereof. 19-62. (canceled)